We propose to develop a human somatic tissue culture model system that can be used to identify and characterize mutations in genes that cause PIDs (primary immune deficiencies). In this application we demonstrate that this approach can be used to provide a structure:function understanding of the DNA repair genes that regulate V(D)J {variable(diversity)joining} recombination, CSR (class switch recombination) and SHM (somatic hypermutation). V(D)J recombination and CSR are site-specific DNA rearrangement processes absolutely required for the development of the human immune system. SHM utilizes a non-essential DNA mutagenesis process that evolution has conscripted in a positive fashion to increase the efficacy of the immune system. Over the past decade it has been demonstrated that patients with mutations in any of about a dozen genes, most of them DNA DSB (double-strand break) repair genes, which are required for either one or all of these processes, result in PIDs when mutated. Importantly, most of these genes have functions outside of the immune system (e.g., signaling, genomic stability, and telomere maintenance) and it is not always clear which human symptoms/pathologies are associated with which activity of the gene, rendering therapeutic intervention difficult and even potentially dangerous. We have developed over the past several years a system using rAAV (recombinant adeno-associated virus) where targeted mutations can be made in virtually any human gene and in virtually any human cell line. We have used this system - in proof-of-principle experiments - to make human cell lines null for a number of PID-causative genes. In addition, we have spent several decades acquiring reagents, assays and expertise in a veritable bevy of areas relevant to DNA repair, V(D)J, CSR, SHM and telomere maintenance. By introducing either wild-type cDNAs or cDNAs containing human mutations into these genetically modified cell lines the impact of patient mutations on each metabolic pathway can be accurately assessed. These experiments should enhance our understanding of the basic domains and activities of these genes. In addition, it is reasonable to anticipate that the information generated from this study (i.e., a correlation of patient mutations to activity) can be productively used in the clinic to treat and/or diagnose PIDs. We believe that our proposal has a number of important general strengths: 1) it can in principle be used for any of the 120+ genes that - when mutated - are known to cause PIDs, 2) it can be used with any human somatic cell line of the researcher's choosing, 3) it allows for a rigorous assessment of whether a naturally occurring sequence variant is an inconsequential polymorphism or a debilitating mutation, 4) it permits a detailed structure:function analysis of the gene under study and 5) it provides novel cell lines that can be used for pre-clinical therapeutic studies. PUBLIC HEALTH RELEVANCE: Project Narrative: Hendrickson Primary immune deficiencies currently affect ~500,000 Americans and newly affected individuals are accruing at the rate of 1 per 1,200 live births. This disease is genetically very heterogeneous with over 120 disease-causing genes correlated with over 200 clinically distinct types of immune deficiencies. Here we propose to develop a human somatic cell culture system in which we can characterize the great bulk of these genes and their relevant activities. The development of such a system has the ability to yield better therapies and is thus clearly relevant to the mission of NIH.)